1. Field of the Invention
The present invention relates to methods of disinfecting articles infected with nanobacteria, and methods of treating patients infected with nanobacteria.
2. Description of the Related Art
The formation of discrete and organized inorganic crystalline structures within macromolecular extracellular matrices is a widespread biological phenomenon generally referred to as biomineralization. Mammalian bone and dental enamel are examples of biomineralization involving apatite minerals. Environmental apatite stones have almost the same chemical composition as in bone and dentine. Recently, bacteria have been implicated as factors in biogeochemical cycles for mineral formation in aqueous sediments. The principal constituent of modern authigenic phosphate minerals in marine sediments is carbonate (hydroxy)fluorapatite Ca10(PO4)6xe2x88x92x(CO3)x(F,OH)2+x. Microorganisms are capable of depositing apatite outside thermodynamic equilibrium in sea water. They can segregate Ca from Mg, and actively nucleate carbonate apatite by means of specific oligopeptides under conditions pH  less than 8.5 and [Mg]:[Ca] greater than 0.1. Such conditions are also present in the human body.
Nanobacteria approach the theoretical limit of the self-replicating life with a size of only one hundredth of that of usual bacteria. Nanobacteria can be isolated from mammalian blood and blood products (see, U.S. Pat. No. 5,135,851 to Kajander, the contents of which are incorporated herein by reference). Energy-dispersive X-ray microanalysis and chemical analysis reveals that nanobacteria produce biogenic apatite on their cell envelope. The thickness of the apatite depends mostly on the culture conditions of the nanobacteria. Nanobacteria are the smallest cell walled, apatite forming bacteria isolated from mammalian blood and blood products. Their small size (0.05-0.5 xcexcm), and unique properties make their detection difficult with conventional microbiological methods. In nanobacteria-infected mammalian cells, electron microscopy revealed intra- and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification.
The present inventors have discovered nanobacteria in human and cow blood that are cytotoxic in vitro and in vivo. They have been deposited in DSM, Braunschweig, Germany at accession No. 5819-5821. Human and bovine nanobacteria grow similarly, share the same surface antigens, and other special features. They both produce carbonate apatite as well. Nanobacteria possess unusual properties making their detection difficult with standard microbiological methods. Although they typically have diameters of 0.2-0.5 xcexcm, they also exist in tiny forms (0.05-0.2 xcexcm) as observed using transmission electron microscopy (TEM). Thus nanobacteria manage to pass through 0.1 xcexcm filters. Nanobacteria are poorly disruptable, stainable, fixable and exceptionally resistant to heat. Their doubling time is about 3 days. High doses of xcex3-irradiation or aminoglycoside antibiotics prevented their multiplication. According to the 16S rRNA gene sequence (EMBL X98418 and X98419), nanobacteria fall within the xcex1-2 subgroup of Proteobacteria, which also includes Brucella and Bartonella species. The latter genera include human and animal pathogens that share similarities with nanobacteria, e.g., some of the same antigens and cytopathic effects.
Competition for nutrients necessary for life is enormous in natural environments and thus clever adaptations and survival strategies for unfavorable conditions are needed. Bacteria can form spores, cysts and biofilm, which help them survive unfavorable periods of time. Bacteria in such forms have significantly slower metabolic functions, but vegetative cells can slow down their metabolism as well. The increased resistance of bacteria in biofilm or as spores is not only because of the slower metabolic rate. The impermeable structures around the organism serve as mechanical barriers blocking the entrance of potentially harmful compounds. Some additional mechanisms are also known which help in the survival of bacteria. The heat resistance of bacterial spores can be attributed to three main factors, these are protoplast dehydration, mineralization and thermal adaptation. Radiation resistance is commonly associated with sophisticated DNA repair systems. Minimizing metabolic rate and multiplication are obviously the main preconditions for bacterial survival, allowing time for the repair of DNA and other damaged cellular components. Very slow metabolism, and ability to form biofilm are also characteristics of nanobacteria. Because of their minimal size, the presence of complicated systems for nucleic acid repair in nanobacteria seems very unlikely. A possible explanation for the observed gamma irradiation resistance may be their very small size, and the peculiarities in their nucleic acid structure.
Apatite may play a key role in the formation of kidney stones. The crystalline components of urinary tract stones are calcium oxalate, calcium phosphate, struvite, purines, or cystine. The majority of urinary stones are admixtures of two or more components, with the primary admixture being calcium oxalate and apatite. Furthermore, fermentor model studies have shown that calcium phosphate nidi are always formed initially, and may subsequently become coated with calcium oxalate or other components. Urinary tract infection, causing struvite and carbonate apatite formation, is a common cause of kidney stones. Conventional therapy has usually consisted of surgical removal of the stone, combined with a short course of antimicrobial therapy. Such treatment is curative in about 50% of cases. Recurrent stone formation and progressive pyelonephritis occur in those who are not cured. The morbidity and expense that result from this disease is significant.
Tissue calcification of carbonate apatite in nature is common in other diseases, e.g., atherosclerotic plaques accumulate calcium phosphate. 25% of atherosclerotic plaques in human aorta specimens were found to contain nanobacterial by immunoassay and immunohistochemical staining. Hemodialysis patients can develop extensive metastatic and tumoral calcification. Acute periarthritis is apatite arthropathy related to intratendinous calcifications. Apatite crystals also cause inflammation when injected into the synovial space. Tissue calcification is also found in several kinds of cancer.
Pulp stones or denticles are polymorphous mineralized bodies of various sizes occasionally found in the pulpal connective tissue of human teeth. Their etiology remains unclear although they have been frequently associated with aging or pathology of the pulp. They may also be present in permanent teeth that are impacted free of pathology for a long time. Although pulp stones have been extensively studied morphologically, their origin is still obscure and little is known about their chemical composition. An histochemical study of pulpal calcifications has shown that the organic matrix consists of reticular connective tissue fibers and a ground substance containing glycoproteins and acid polysaccharides. The mineral phase of pulp calcification has been studied with X-ray energy dispersive spectrometry and chemical analysis, and proven that calcium salts are deposited in the form of apatite, possibly carbonate containing apatite. In fact, there is not much difference between the chemical structure of a tooth and denticles. Bone and tooth formation in the body have similar mechanisms, leaving many unanswered questions. Apatite formation in the body (except in tooth and bone) is called pathologic biomineralization, e.g., dental pulp stones, kidney stones, and joint calcifications.
Malacoplakia is a rare chronic inflammatory disease of unknown cause, but a bacterial factor has been strongly implicated. It may be fatal. The disease is characterized by von Kossa staining positive, calcified laminated or target-shaped bodies termed Michaelis-Gutmann bodies which are composed of apatite. The structure of these calcospherules closely resembles calcified nanobacteria.
Tissue calcifications are found in several diseases such as ovarian serous tumor, papillary adenocarcinoma of the endometrium, breast carcinoma, papillary carcinoma of the thyroid, duodenal carcinoid tumor, and craniopharyngioma. In many malignant tumors, needle-shaped crystals are found in epithelial cells. To detect this kind of calcification it is necessary to use electron microscopy, since the crystals are too small to be seen with the light microscope, and their origin is unknown. Many malignant cells have receptors for nanobacterial adherence. They could introduce nanobacteria into the tumor with subsequent calcification. Furthermore, some dividing cells under inflammatory stimuli may have receptors for adherence, e.g., in atherosclerotic plaques known to have calcium phosphate accumulation. In this disease, although electron probe analysis showed that the surface and interior of the mineral deposit had the same chemical composition, SEM revealed different kinds of structures such as spherical particles and fibers which resemble nanobacteria. Similarly, acute periarthritis has been associated with the presence of hydroxyapatite crystals in the joints.
Alzheimer plaques may be labeled with anti-nanobacterial polyclonal antibodies. These polyclonal antibodies contain some autoantibodies, and the present inventors have also obtained some monoclonal autoantibodies in nanobacterial immunizations. Slow bacterial infection has been suggested to play a role in autoimmune diseases. Tissue calcification is often present in these diseases. Nanobacteria are a new example of slowly growing organisms, infecting man for long periods of time. The apatite structure and anomalous nucleic acids may contribute to abnormalities in immune response to this infection.
Several aspects of biogenic apatite nucleation, crystal growth and morphology have been determined both in vivo and in vitro. However, many details remain unresolved, including the specific nature of the initial precipitating phases, the mechanism and factors which control the incorporation of ionic impurities into the crystal lattice, details of the crystallographic ultrastructure and morphology in mineralized tissues (bone, dentine), and the relationship of the inorganic components with the complex collagen based matrix. The reason behind the calcium phosphate deposition in many diseases remain speculative. It has been shown that an accumulation of calcium in mitochondria, which is presumably dependent upon residual substrate for energy production, appeared to cause calcification. Amorphous calcium phosphate in the form of spheroids, and possibly fine fibrils and granules, also appears to play a role in calcification by their transformation into apatite.
The present invention provides methods for sterilizing articles contaminated with nanobacteria. Such methods according to the present invention will be particularly useful for disinfecting and/or sterilizing medical equipment and solutions used in patient treatment and diagnosis.
The present invention also provides methods of preventing nanobacterial infection, and treating patients infected with nanobacteria. In particular, the present invention provides a method for preventing the recurrence of kidney stones in a patient that has suffered from kidney stones, comprising administration of an antibiotic in an amount effective to inhibit or prevent the growth and development of nanobacteria.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.